This invention was made with government support under R01HL40411, HL43344 and RR04870 awarded by the National Institutes of Health. The government has certain rights in the invention.
1. Field of the Invention
This invention relates to a new method for the treatment or prevention of disease states associated with elevated levels of homocysteine, comprising administering a pharmaceutical composition containing a nitrosating compound to a patient.
2. Brief Description of the Background Art
Homocysteine is a thiol-containing amino acid that results from the demethylation of methionine. Homocysteine is readily oxidized in body fluids to homocysteine disulfide (homocystine), mixed disulfides, and the cyclized oxidation product, homocysteine thiolactone (HTL).
Homocystinurias are clinical disorders which result from a number of different inborn or acquired defects in the pathway which control homocysteine metabolism. These disorders are characterized by increased concentrations of homocystine in the blood and urine.
The most common form of homocystinuria results from a deficiency of cystathionine .beta.-synthase, an enzyme in the transsulfuration pathway by which methionine is converted to cysteine. Another form results from a deficiency of 5,10-methyl tetrahydrofolate reductase, which provides substrate for the B.sub.12 -dependent conversion of homocysteine to methionine. Homocystinurias also result from deficiencies of necessary metabolic cofactors, such as folate, and vitamins B.sub.12 and B.sub.6. In addition, homocystinuria may result from bile acid sequestrant plus niacin therapy which impairs folate absorption. Finally, these disorders may also be secondary to abnormal kidney function, or to other mechanisms not yet characterized.
In some patients, metabolic defects may cause elevations of homocysteine in serum which are not high enough to cause the excretion of detectable amounts of homocystine in the urine, yet result in serious pathological consequences. Thus, investigators have suggested the term "hyperhomocysteinemia" to refer to a transient or persistent elevation of serum homocysteine, which may or may not be accompanied by increased homocystine in the urine (Malinow et al. Circulation 79:1180-1188 (1989)).
Hyperhomocysteinemia causes a variety of disease states which manifest in serious vascular, ocular, neurological, and skeletal abnormalities.
Atherogenesis and thrombosis are well-recognized complications of hyperhomocysteinemia. (Clarke et al., N. Engl. J. Med. 324:1149-1155 (1991); Malinow, Circulation 81:2004-2006 (1990)). Afflicted individuals often experience serious thrombotic complications at an early age, with thrombosis of extracranial and intracranial arteries, veins and sinuses, as well as coronary occlusion, being common fatal occurrences. In young patients, occlusion of peripheral arteries often results in renal vascular hypertension, intermittent claudication or mesenteric ischemia, and thrombosis may be followed by pulmonary embolism. If left untreated, juvenile hyperhomocysteinemia results in the death of more than 50% of the affected individuals before the age or 20, due to myocardial infarction, stroke or pulmonary embolism (Brattstrom, L. E. et al., Metabolism 34(11):1073-1077 (1985)). In older patients, moderate hyperhomocysteinemia is found to exist in 20-30% of those afflicted with coronary and peripheral vascular disease (Malinow, M. R., Circulation 81:2004-2006 (1990)).
Normal hemostasis is a dynamic process in which platelet aggregates form and disperse continuously. To achieve this process, physiologic stimuli for platelet activation are counterbalanced by vascular endothelial cell secretion of such substances as prostacyclin and endothelium-derived relaxing factor (EDRF), which has been identified as nitric oxide (NO), or a closely related derivative thereof (Palmer et al., Nature 327:524-526 (1987); Ignarro et al., Proc. Natl. Acad. Sci., USA 84:9265-9269 (1987)). Accordingly, endothelial dysfunction or injury may predispose an individual to thrombosis and vascular occlusive events.
Inferential evidence has suggested the existence of a causal mechanism for endothelial dysfunction and enhanced platelet aggregation in hyperhomocysteinemia. Marked platelet accumulation at sites of vascular injury and platelet-rich occlusive thrombi are distinctive pathological features of both human and experimental hyperhomocysteinemia (James, JACC 15:763-774 (1990); Harker et al., N. Engl. J. Med. 291:537-543 (1974); Harker et al., J. Clin. Invest. 58:731-741 (1976)). To explain this pathologic appearance, several groups have reported direct pro-aggregatory effects of homocysteine and HTL (McDonald et al., Lancet 1:745-746 (1964); Graeber et al., Pediatr. Res. 16:490-493 (1982); McCully et al., Res. Comm. Chem. Path. Pharm. 56(3):349-360 (1987)). However, these pro-aggregatory actions have not been demonstrated with uniformity, and supportive biochemical and molecular mechanisms have not been well elucidated. Moreover, the platelet-activating effects of homocysteine, attributed to its reactive SH group, are difficult to reconcile with the known anti-platelet properties of other biological thiols with similar chemical and physical characteristics. For example, it has been shown that glutathione, cysteine, and N-acetylcysteine have anti-platelet effects, at millimolar concentrations (Thomas et al., Thromb Res. 44:859-866 (1986); Stamler et al., Am. J. Cardiol. 62:377-380 (1988)).
Other investigators have suggested that homocysteine-induced endothelial injury, by exposing sub-endothelial connective tissue, represents an alternative mechanism for platelet activation in vivo (Harker et al., N. Engl. J. Med. 291:537-543 (1974); Harker et al., J. Clin. Invest. 58:731-741 (1976)). Endothelial toxicity has subsequently been confirmed, and attributed to H.sub.2 O.sub.2, generated by way of the SH group, or to the direct toxic effects of HTL (Wall et al., Thromb. Res. 18:113-121 (1980); Starkebaum et al., J. Clin. Invest. 77:1370-1376 (1986); McCully et al., Am. J. Path. 61(1):1-8 (1970)). In addition, high levels of oxidation products of homocysteine may further significantly increase the homocysteine-related burden in plasma and the cell cytosol, and thereby contribute to its pathogenicity (Kang et al., Am. Soc. Clin. Invest. 77:1482-1486 (1986); McCully, Nature (London) 231:391-92 (1971)). Others have proposed that homocysteine potentiates the auto-oxidation of low-density lipoprotein cholesterol and promotes thrombosis through enhanced platelet aggregation.
In addition to its thrombogenic and atherogenic effects, homocysteine interferes with the normal cross-linking of collagen. This effect is responsible, not only for the vascular, but also for the ocular, skeletal and neurological complications of hyperhomocysteinemia. For example, altered collagen in the suspensory ligament of the optic lens causes dislocated lenses (ectopia lentis), and in the bone matrix, results in osteoporosis.
Neurological complications of homocysteine include delayed psychomotor development, severe mental retardation, seizures, and upper motor neuron dysfunction. While recurrent cerebrovascular accidents secondary to thrombotic disorders may be responsible for the mental retardation and other neurological complications, direct chemical cytotoxic effects on cerebral cell metabolism have also been implicated. Furthermore, widespread pathological changes in the central nervous system, liver, kidneys and skeletal muscles observed in some hyperhomocysteinemia patients have been attributed to a direct cytotoxic effect exerted by homocysteine.
In addition to the adverse effects attributed to homocysteine, other sulfur-containing amino acids, such as cysteine, have also been associated with vascular and connective tissue disorders. It has recently been appreciated that abnormalities in the oxidative metabolism of cysteine are found in rheumatoid arthritis and systemic lupus. These disease states are also associated with vasculitis (Gordan et al., Lancet 339:25-26 (1992)).
Currently available methods for treating hyperhomocysteinemia consist essentially of administration of vitamin supplements, such as pyridoxine, folate, choline, betaine, or cobalamin, in an attempt to reduce serum homocysteine levels. A few afflicted infants, diagnosed in the newborn period, have been treated successfully with methionine-restricted, cystine-supplemented diets. However, the success of this method depends on accurate diagnosis within the newborn period, and accounts for a very small number of those patients afflicted with hyperhomocysteinemia.
Vitamin therapy has been shown to cause a decrease in plasma homocysteine levels in some patients; however, numerous patients afflicted with hyperhomocysteinemia experience little or no reduction in homocysteine levels as a result of this therapy. Furthermore, vitamin therapy does not directly counteract the toxic effects of homocysteine, or provide a means to ameliorate the pathological effects resulting from past exposure to homocysteine. Consequently, there is no evidence that current modes of therapy reduce the risk of atherothrombotic (or other) complications in afflicted adults.
Successful treatment of hyperhomocysteinemia depends, not only on the immediate reduction of homocysteine levels, but more importantly, on directly counteracting the toxic effect produced by homocysteine. Therefore, a clinical need exists for a pharmacological method which directly counteracts the immediate toxicity of homocysteine and also ameliorates the pathophysiological abnormalities resulting from past exposure to homocysteine.